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MicroRNA 195-5p Targets Foxo3 Promoter Region to Regulate Its Expression in Granulosa Cells.

ABSTRACT: Forkhead box O3 (Foxo3) is a member of the FOXO subfamily within the forkhead box (FOX) family, which has been shown to be essential for ovarian follicular development and maturation. Previous studies have shown the abundant expression of miR-195-5p in the nuclei of porcine granulosa cells (GCs), suggesting its potential role during ovarian follicle growth. In this study, a conditional immortalized porcine granulosa cell (CIPGC) line was used to determine whether the expression of Foxo3 could be regulated by the nuclear-enriched miR-195-5p. Through silico target prediction, we identified a potential binding site of miR-195-5p within the Foxo3 promoter. The over-expression of miR-195-5p increased Foxo3 expression at both mRNA and protein levels, while the knockdown of miR-195-5p decreased the expression of Foxo3. Furthermore, driven by the Foxo3 promoter, luciferase reporter activity was increased in response to miR-195-5p, while the mutation of the miR-195-5p binding site in the promoter region abolished this effect. In addition, the siRNA knockdown of Argonaute (AGO) 2, but not AGO1, significantly decreased Foxo3 transcript level. However, miR-195-5p failed to upregulate Foxo3 expression when AGO2 was knocked down. Moreover, chromatin immunoprecipitation (CHIP) assay showed that anti-AGO2 antibody pulled down both AGO2 and the Foxo3 promoter sequence, suggesting that AGO2 may be required for miR-195-5p to regulate Foxo3 expression in the nucleus. Additionally, Foxo3 expression was significantly increased by valproic acid (VPA), the inhibitor of deacetylase, as well as by methyltransferase inhibitor BIX-01294, indicating the involvement of histone modification. These effects were further enhanced in the presence of miR-195-5p and were decreased when miR-195-5p was knocked down. Overall, our results suggest that nuclear-enriched miR-195-5p regulates Foxo3 expression, which may be associated with AGO2 recruitment, as well as histone demethylation and acetylation in ovarian granulosa cells.

SUBMITTER: Bai Y

PROVIDER: S-EPMC8267755 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEsophageal cancer is a high incident cancer worldwide with poor survival and limited therapeutic options. Alterations of microRNAs are common in cancers, and many of these micro RNAs are potential therapeutic and diagnostic targets to treat these cancers. miR-10b-3p located in chromosome region 2q31.1, and its expression is frequently increased in esophageal squamous cell carcinoma (ESCC). However, the biological functions, clinical significance and therapeutic implications of miR-10b-3p in ESCC remain unclear.MethodsThe expression levels of miR-10b-3p in ESCC specimens were analyzed by in situ hybridization (ISH) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. Ectopic overexpression of miR-10b-3p in ESCC cells, mouse xenograft model, and metastasis model were used to evaluate the effects of miR-10b-3p on proliferation, and migration of cancer cells. Luciferase reporter assay and Western blot were performed to validate the potential targets of miR-10b-3p after the preliminary screening by computer-aided microarray analysis.ResultsWe found that miR-10b-3p expression levels were significantly upregulated in the tumor tissues and serum samples of patients with ESCC. The expression levels of miR-10b-3p in both tumor tissues and serum samples were inversely associated with lymph node metastasis and clinical stages. We identified the expression level of miR-10b-3p in ESCC cancer samples as an independent prognostic marker of the overall survival rates of ESCC patients. We found more frequent hypomethylation of the CpG sites located upstream of the miR-10b-3p gene in the ESCC tissues compared with in the adjacent normal tissues, and the DNA methylation status of miR-10b-3p promoter region inversely correlated with the expression levels of miR-10b-3p. Ectopic overexpression of miR-10b-3p promoted cell proliferation, colony formation, migration and invasion in ESCC. While knockdown of miR-10b-3p had the opposite effects, particularly in promoting apoptosis. Mouse xenograft model confirmed that miR-10b-3p functions as a potent oncogenic miRNA in ESCC, which also promoting ESCC metastasis. Mechanistically, we found miR-10b-3p regulated FOXO3 expression by directly binding to the 3'-untranslated region. And systemic delivery of miR-10b-3p antagomir reduced tumor growth and inhibit FOXO3 protein expression in nude mice.ConclusionsCollectively, our findings suggested upregulated expression of miR-10b-3p caused by promoter hypomethylation contributed to the progression of ESCC; Thus, miR-10b-3p is a potentially effective biomarker for ESCC that could have further therapeutic implications.

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MicroRNA 195-5p Targets Foxo3 Promoter Region to Regulate Its Expression in Granulosa Cells.

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